The Nature of Genes
Early ideas to explain how genes work came from studying human diseases. |
Archibald Garrod proposed that patients with the disease alkaptonuria lacked a particular enzyme. |
Beadle and Tatum studied Neurospora crassa.
They looked for fungal cells lacking specific enzymes. |
Beadle and Tatum results was that each mutated enzyme disrupted one key enzyme in the metabolic pathway. |
Prokaryotic Transcription
Prokaryotic Transcription: Single RNA polymerase
- Initiation of mRNA synthesis does not require a primer |
Prokaryotic Transcription requires a Promoter, Start Site, and a termination site. |
Transcription occurs in three major stages:
- Initiation
- Elongation
- Termination |
Initiation: RNA polymerase binds to the promoter |
Promoter: Forms a recognition and binding site for the RNA polymerase.
- Found upstream of the start site.
- Not transcribed.
- Asymetrical: indicate site of initiation and direction of termination. |
Elongation: RNA transcript grows in the 5'-to-3' direction as ribonucleotides are added.
- Transcription bubble: contains RNA polymerase, DNA template, and growing RNA transcript.
- After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble. |
Termination: Marked by sequence that signals "stop" to polymerase.
- Causes the formation of phosphodiester bonds to cease.
- RNA-DNA hybrid within the transcription bubble dissociates.
- RNA polymerase releases the DNA.
- DNA rewinds. |
Hairpin in RNA causes RNA polymerase to pause |
U:A base pairs weaken the DNA/RNA bonding. |
Prokaryotic transcription is coupled to translation
- mRNA begins to be translated before transcription is finished. |
Frameshift mutations
- Addition or deletion of a single base
- Much more profound consequences
- Alter reading frame downstream
- Triplet repeat expansion mutation
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Hunting disease
Repeat unit is expanded in the disease allele relative to the normal |
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Transcription and Translation
The Central Dogma |
Information only flows from: DNA-->RNA-->protein
First described by Francis Crick. |
Transcription |
DNA--> RNA
- DNA-directed synthesis of RNA
- Only template strand of DNA used
- T in DNA replaced by U in RNA.
- mRNA used to direct synthesis of polypeptides. |
Translation |
- Synthesis of polypeptides.
- Takes place at ribosome.
- Requires several kinds of RNA. |
RNA |
All synthesized from DNA template by transcription
- Messenger RNA (mRNA).
- Ribosomal RNA (rRNA).
- Transfer RNA (tRNA).
- Small nuclear RNA (snRNA)
- Signal recognition particle RNA (SRP RNA).
- Micro-RNA (miRNA). |
Eukaryotic Transcription
3 different RNA polymerase!! |
RNA polymerase I: Transcribes rRNA. |
RNA polymerase II: treanscribes mRNA and some snRNA. |
RNA polymerase III: transcribes tRNA and some other small RNAs |
Each RNA polymerase recognizes it own promoter. |
Initiation of transcription: Requires a series of transcrption factors (helper).
- Transcription factors: Necessary to get the RNA polymerase II enzyme to a promoter* and to initiate gene expression. |
Elongation: RNA transcribed from the DNA template. |
Termination not as well defined. |
Initiation of trancription
- Transcription factors bind to a promoter region and recruit RNA polymerase.
- Forms the initation complex.
Protein Targeting
In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER). |
Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition (SRP). |
- The signal sequence and SRP are recognized by RER receptor proteins
- Docking holds ribosome to RER
- Beginning of the protein-trafficking pathway
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Mutation: Altered Genes
Point mutations |
alter a single base |
Base substitution |
substitute one base for another |
Siletn mutation |
same amino acid inserted |
Missense mutation |
changes amino acid inserted
- Transitions
- Transversions |
Nonsense mutations |
changed to stop codon |
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The Genetic Code
Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order.
- They introduced single nucleotide insertions or deletions and looked for mutations (Frameshift mutations) |
A Codon is a block of three DNA nucleotides corresponding to an amino acid, |
Spaced Codons: Codon sequence in a gene punctuated. |
Unspaced Codons: codons adjacent to each other.
- Marshall Nirenberg identified the codons that specify each amino acid. |
Stop Codon: 3 codons (UAA, UGA, UAG) used to terminate translation |
Start Codon: Codon (AUG) used to signify the start of translation |
Code is degenerate: Some amino acids are specified by more that one codon. |
Code practically unicersal: Strongest evidence that all living things share common ancestry.
- Advanced in genetic engineering.
- Mitochondria and cloroplast have some differences in "stop" signals. |
mRNA modifications
In eukaryotes the primary transcript must be modified to become mature mRNA |
Addition of a 5' cap Protects nucleotides from getting lost, from degradation.
- Involved in translation initiation. |
Addition of a 3' poly-A tail Created by poly-A polymerase, protection from degradation
- Puts whole string of A's (AAA) to protect! |
Removal of noncoding sequences (introns): Pre-mRNA splicing done by spliceosome.
- Cut it out to get rid of it!!! |
tRNA charging reaction
Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs. |
Charged tRNA has an amino acid added using the energy from ATP.
-Can undergo peptide bond formation without additional energy. |
Ribosomes do not verify amino acid attached to tRNA. |
The ribosome has multiple tRNA binding sites: |
P site: binds the tRNA attached to the growing peptide chain |
A site: binds the tRNA carrying the next amino acid. |
E site: binds the tRNA that carried the last amino acid, tRNA exits ribosome. |
The ribosome has two primary functions
- Decode the mRNA.
- Form peptide bonds. |
Peptidyl transferase:
- Enzymatic component of the ribosome.
- Forms peptide bonds between amino acids. |
Chromosomal mutations
Chang the structure of a chromosome |
Deletions: part of chromosome is lost |
Duplication: part of chromosome is copied |
Inversion: part of chromosome in reverse order |
Translocation: part of chromosome is moved to a new location |
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Eukaryotic pre-mRNA splicing
Introns |
non-coding sequences |
Exons |
sequences that will be translated |
Small ribonucleoprotein particles (snRNPs "snurps") |
Looks for introns and exons and recognizes it. |
Spliceosomes |
responsible for removing introns |
snRNPs cluster with other proteins to form spliceosome
tRNA and Ribosomes
tRNA moleules carry amino acids to the ribosome for incorporation into a polypeptide.
- Aminoacyl-tRNA synthetase add amino acids to the acceptor stem of tRNA.
- Anticodon loop contains 3 nucleotides complementary to mRNA codons. |
Translation
Process by which the mRNA transcript is read by the ribosomes and used to make a polypeptide.
Occurs in 3 main stages:
- Initiation
- Elongation
- Termination |
There are some important differences between translation in prokaryotes and eukaryotes. |
In prokaryotes, initiation complex includes Initiator tRNA charged with N-formylmethionine {[nl}}- Small ribosomal subunit
- mRNA strand |
- Ribosome binding sequence (RBS) of mRNA positions small subunit correctly.
- Large subunit now added.
- Initiator tRNA bound to P site with A site empty.
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Initiations in eukaryotes similar except:
- Initiating amino acid is methionine.
- Lack of an RBS – small subunit binds to 5′ cap of mRNA. |
Elongation adds amino acids
- 2nd charged tRNA can bind to empty A site
- Requires elongation factor called EF-Tu to bind to tRNA and GTP
- Peptide bond can then form.
- Addition of successive amino acids occurs as a cycle. |
- There are fewer tRNAs than codons
- Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon
- This allows fewer tRNAs to accommodate all codons
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Termination
- Elongation continues until the ribosome encounters a stop codon
- Stop codons are recognized by release factors which release the polypeptide from the ribosome |
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